A major problem in cancer chemotherapy remains the development of tumor cell resistance to multiple chemotherapeutic agents. Overexpression of the multidrug resistance gene (MDR1) leads to the appearance of P- glycoprotein (P-gp) in the plasma membrane of tumor cells. This protein functions as a drug efflux pump resulting in tumor cell resistance to many cytotoxic drugs. The long-term objectives of this grant proposal are (1) to further understand the structural determinants and biochemical characteristics of P-gp, and (2) to unravel the molecular mechanism by which P-gp can recognize a wide variety of lipophilic agents including both cytotoxic drugs and MDR modulators. The specific aims of this proposal are to (1) synthesize specific photoaffinity probes, (2) investigate the modes of drug interaction with P-gp, (3) identify drug binding domains of P-gp, and (4) purify drug binding fragments of P-gp and identify the specific amino acid sequences of the drug binding site(s). These studies will increase our knowledge of the biochemistry and molecular nature of P-gp and could provide a framework for the rational design and synthesis of effective MDR modulators.